The present invention relates to mammalian mahogany genes, including the human mahogany gene, which are novel genes involved in the control of mammalian body weight. The invention encompasses nucleotide sequences of the mahogany gene, host cell expression systems of the mahogany gene, and hosts which have been transformed by these expression systems, including transgenic animals. The invention also encompasses novel mahogany gene products, including mahogany proteins, polypeptides and peptides containing amino acid sequences mahogany proteins, fusion proteins of mahogany proteins polypeptides and peptides, and antibodies directed against such mahogany gene products.
The present invention also relates to methods and compositions for the diagnosis and treatment of mammalian body weight disorders, including obesity, cachexia, and anorexia, and for the identification of subjects susceptible to such disorders. Further, the invention relates to methods of using the mahogany gene and gene products of the invention for the identification of compounds which modulate the expression of the mahogany gene and/or the activity of the mahogany gene product. Such compounds can be useful as therapeutic agents in the treatment of mammalian body weight disorders, including obesity, cachexia, and anorexia.
Obesity represents the most prevalent of body weight disorders, and it is the most important nutritional disorder in the western world, with estimates of its prevalence ranging from 30% to 50% within the middle-aged population. Other body weight disorders, such as anorexia nervosa and bulimia nervosa, which together affect approximately 0.2% of the female population of the western world, also pose serious health threats. Further, such disorders as anorexia and cachexia (wasting) are also prominent features of other diseases such as cancer, cystic fibrosis, and AIDS.
Obesity, defined as an excess of body fat relative to lean body mass, also contributes to other diseases. For example, this disorder is responsible for increased incidence of diseases such as coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia, and some cancers (See, e.g., Nishina, P. M. et al., 1994, Metab. 43: 554-558; Grundy, S. M. and Barnett, J. P., 1990, Dis. Mon. 36: 641-731). Obesity is not merely a behavioral problem, i.e., the result of voluntary hyperphagia. Rather, the differential body composition observed between obese and normal subjects results from differences in both metabolism and neurologic/metabolic interactions. These differences seem to be, to some extent, due to differences in gene expression, and/or level of gene products or activity (Friedman, J. M. et al., 1991, Mammalian Gene 1: 130-144).
The epidemiology of obesity strongly shows that the disorder exhibits inherited characteristics (Stunkard, 1990, N. Eng. J. Med. 322: 1438). Moll et al. have reported that, in many populations, obesity seems to be controlled by a few genetic loci (Moll et al., 1991, Am. J. Hum. Gen. 49: 1243). In addition, human twin studies strongly suggest a substantial genetic basis in the control of body weight, with estimates of heritability of 80-90% (Simopoulos, A. P. and Childs, B., eds., 1989, in xe2x80x9cGenetic Variation and Nutrition in Obesityxe2x80x9d, World Review of Nutrition and Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976, Acta. Paediatr. Scand. 65: 279-287).
In other studies, non-obese persons who deliberately attempted to gain weight by systematically over-eating were found to be more resistant to such weight gain and able to maintain an elevated weight only by very high caloric intake. In contrast, spontaneously obese individuals are able to maintain their status with normal or only moderately elevated caloric intake. In addition, it is a commonplace experience in animal husbandry that different strains of swine, cattle, etc., have different predispositions to obesity. Studies of the genetics of human obesity, and of animal models of obesity demonstrate that obesity results from complex defective regulation of both food intake, food induced energy expenditure, and of the balance between lipid and lean body anabolism.
There are a number of genetic diseases in man and other species which feature obesity among their more prominent symptoms, along with, frequently, dysmorphic features and mental retardation. For example, Prader-Willi syndrome (PWS; reviewed in Knoll, J. H. et al., 1993, Am. J. Med. Genet. 46: 2-6) affects approximately 1 in 20,000 live births, and involves poor neonatal muscle tone, facial and genital deformities, and generally obesity.
In addition to PWS, many other pleiotropic syndromes have been characterized which include obesity as a symptom. These syndromes are genetically straightforward, and appear to involve autosomal recessive alleles. Such diseases include, among others, Ahlstroem, Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.
A number of models exists for the study of obesity (see, e.g., Bray, G. A., 1992, Prog. Brain Res. 93: 333-341; and Bray, G. A., 1989, Amer. J. Clin. Nutr. 5: 891-902). For example, animals having mutations which lead to syndromes that include obesity symptoms have also been identified. Attempts have been made to utilize such animals as models for the study of obesity, and the best studied animal models to date for genetic obesity are mice. For reviews, see, e.g., Friedman, J. M. et al., 1991, Mamm. Gen. 1: 130-144; Friedman, J. M. and Liebel, R. L., 1992, Cell 69: 217-220.
Studies utilizing mice have confirmed that obesity is a very complex trait with a high degree of heritability. Mutations at a number of loci have been identified which lead to obese phenotypes. These include the autosomal recessive mutations obese (ob), diabetes (db), fat (fat), and tubby (tub).
The dominant Yellow mutation (Ay) at the agouti locus causes a pleiotropic syndrome which causes moderate adult onset obesity, a yellow coat color, and a high incidence of tumor formation (Herberg, L. and Coleman, D. L., 1977, Metabolism 26:59), and an abnormal anatomic distribution of body fat (Coleman, D. L., 1978, Diabetologia 14:141-148). The mutation causes the widespread expression of a protein which is normally seen only in neonatal skin (Michaud, E. J. et al., 1994, Genes Devel. 8:1463-1472). The agouti protein has been reported to be a competitive antagonist of xcex1-MSH binding to the melanocortin receptors MC1-R and MC4-R in vitro (Lu et al., 1996, Nature 371:799-802), and the authors speculated that de-regulated ubiquitous expression of agouti may lead to obesity by antagonism of melanocortin receptors expressed outside the hair follicles.
Mahogany (mg) and mahoganoid (md) are mutations that suppress the phenotypic effects of agouti protein in vivo (Lane and Green, 1960, J. Hered. 51: 228-230). The mahogany and mahoganoid mutation have been-mapped to mouse chromosomes 2 and 16, respectively (Green, 1989, xe2x80x9cCatalog of mutant genes and polymorphic locixe2x80x9d, pp. 12-403 in Genetic Variants and Strains of the Laboratory Mouse, Lyon, M. F. and Searle, A. G., eds., Oxford University Press, Oxford). Mutations of both mg and md have been shown to suppress the effects of agouti on obesity as well as on coat color (Miller et al., 1997, Genetics 146: 1407-1415).
In summary, therefore, obesity, which poses a major, worldwide health problem, represents a complex, highly heritable trait. Given the severity, prevalence, and potential heterogeneity of such disorders, there exists a great need for the identification of those genes that participate in the control of body weight.
The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules that are involved in the control of mammalian body weight, and which, further, are associated with mammalian body weight disorders such as obesity, cachexia, and anorexia. The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian mahogany gene, including the human mahogany gene.
In particular, the compositions of the present invention include nucleic acid molecules which comprise the following sequences: (a) nucleotide sequences of the mahogany gene, including, e.g., murine mahogany sequences as shown in FIGS. 2A, 3B-D, 6A-B, 8A, and 9A, as well as allelic variants and homologs thereof, and human mahogany sequences, as shown, e.g., in FIGS. 10A, 18A, 19A and 20A, as well as allelic variants and homologs thereof; (b) nucleotide sequences that encode the mahogany gene product amino acid sequences, as shown, e.g., in in FIGS. 2B, 8B, 9B, 10B, 17, 18B, 19B and 20B; (c) nucleotide sequences that encode portions of the mahogany gene product corresponding to its functional domains and individual exons; (d) nucleotide sequences comprising the novel mahogany gene sequences disclosed herein that encode mutants of the mahogany gene product in which all or a part of one or more of the domains is deleted or altered, as shown, e.g., in FIG. 6; (e) nucleotide sequences that encode fusion proteins comprising the mahogany gene product, or one or more of its domains fused to a heterologous polypeptide; (f) nucleotide sequences within the mahogany gene, as well as chromosome sequences flanking the mahogany gene, see, e.g., FIG. 3, which can be utilized as part of the methods of the present invention for the diagnosis of mammalian body weight disorders, including obesity, cachexia, and anorexia, which are mediated by the mahogany gene, as well as for the identification of subjects susceptible to such disorders; (g) nucleic acid sequences that hybridize to the above described sequences under stringent or moderately stringent conditions, particularly human mg homologs. The nucleic acid molecules of the invention include, but are not limited to, cDNA and genomic DNA sequences of the mahogany gene.
The present invention also encompasses expression products of the nucleic acid molecules listed above; i.e., proteins and/or polypeptides that are encoded by the above mahogany nucleic acid molecules.
Agonists and antagonists of the mahogany gene and/or gene product are also included in the present invention. Such agonists and antagonists will include, for example, small molecules, large molecules, and antibodies directed against the mahogany gene product. Agonists and antagonists of the invention also include nucleotide sequences, such as antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs, that can be used to inhibit or enhance expression of the mahogany gene.
The present invention further encompasses cloning vectors, including expression vectors, that contain the nucleic acid molecules of the invention and can be used to express those nucleic acid molecules in host organisms. The present invention also relates to host cells engineered to contain and/or express the nucleic acid molecules of the invention. Further, host organisms which have been transformed with these nucleic acid molecules are also encompassed in the present invention. Host organisms of the invention include organisms transformed with the cloning vectors described above, e.g., transgenic animals, particularly non-human transgenic animals, and particularly transgenic non-human mammals.
The transgenic animals of the invention include animals that express a mutant variant or polymorphism of a mahogany gene, particularly a mutant variant or polymorphism of a mahogany gene that is associated with a weight disorder such as obesity, cachexia, or anorexia. The transgenic animals of the invention further include those that express a mahogany transgene at higher or lower levels than normal. The transgenic animals of the invention further include those which express the mahogany gene in all their cells, xe2x80x9cmosaicxe2x80x9d animals which express the mahogany gene in only some of their cells, and those in which the mahogany gene is selectively introduced into and expressed in a specific cell type(s). The transgenic animals of the invention also include xe2x80x9cknock-outxe2x80x9d animals. Knock-out animals comprise animals which have been engineered to no longer express the mahogany gene.
The present invention also relates to methods and compositions for the diagnosis of mammalian body weight disorders, including obesity, cachexia, and anorexia, as well as for the identification of subjects susceptible to such disorders. Such methods comprise, for example, measuring expression of the mahogany gene in a patient sample, or detecting a mutation in the mahogany gene in the genome of a mammal, including a human, suspected of exhibiting such a weight disorder. The nucleic acid molecules of the invention can also be used as diagnostic hybridization probes, or as primers for diagnostic PCR analysis to identify of mahogany gene mutations, allelic variations, or regulatory defects, such as defects in the expression of the mahogany gene. Such diagnostic PCR analyses can be used to diagnose individuals with a body weight disorder associated with a particular mahogany gene mutation, allelic variation, or regulatory defect. Such diagnostic PCR analyses can also be used to identify individuals susceptible to such body weight disorders and hyperphagia.
Methods and compositions, including pharmaceutical compositions, for the treatment of body weight disorders such as obesity, cachexia, and anorexia are also included in the invention. Such methods and compositions are capable of modulating the level of mahogany gene expression and/or the level of activity of the mahogany gene product. Such methods include, for example, modulating the expression of the mahogany gene and/or the activity of the mahogany gene product for the treatment of a body weight disorder which is mediated by some other gene, for example by the agouti gene.
The invention still further relates to methods for identifying compounds which modulate the expression of the mammalian mahogany gene and/or the synthesis or activity of mammalian mahogany gene products. Such compounds include therapeutic compounds which can be used as pharmaceutical compositions to reduce or eliminate the symptoms of mammalian body weight disorders such as obesity, cachexia, and anorexia. Cellular and non-cellular assays are described that can be used to identify compounds that interact with the mahogany gene and/or gene product, e.g., modulate the activity of the mahogany gene and/or bind to the mahogany gene product. Such cell-based assays of the invention utilize cells, cell lines, or engineered cells or cell lines that express the mahogany gene product.
In one embodiment, such methods comprise contacting a compound to a cell that expresses a mahogany gene, measuring the level of mahogany gene expression, gene product expression, or gene product activity, and comparing this level to the level of mahogany gene expression, gene product expression, or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound that modulates the expression of the mammalian mahogany gene and/or the synthesis or activity of mammalian mahogany gene products has been identified.
In an alternative embodiment, such methods comprise administering a compound to a host, e.g., a transgenic animal that expresses a mahogany transgene or a mutant mahogany transgene, and measuring the level of mahogany gene expression, gene product expression, or gene product activity. The measured level is compared to the level of mahogany gene expression, gene product expression, or gene product activity in a host that is not exposed to the compound, such that if the level obtained when the host is exposed to the compound differs from that obtained when the host is not exposed to the compound, a compound that modulates the expression of the mammalian mahogany gene and/or the synthesis or activity of mammalian mahogany gene products, and/or the symptoms of a mammalian body weight disorder, such as obesity, cachexia, or anorexia, has been identified.
The Example presented in Section 6, below, describes the genetic and physical mapping of the mahogany gene to a specific 700 kb interval of mouse chromosome 2. The example presented in Section 7, below, describes the identification of a transcription unit within this chromosome interval, referred to herein as the MG gene, which represents the mahogany gene. The expression and sequence analysis of this candidate mahogany gene is described in the example presented in Section 8, below. These experiments prove that the candidate gene MG is indeed the mahogany gene. The example presented in Section 9, below, presents data demonstrating that the mechanism of mahogany action is specific for diet-induced obestity, therefore supporting the use of mahogany antagonists as a specific therapeutic for treatment of diet-induced body weight disorders. The example presented in Section 10, below, presents the identification and characterization of the human mg gene, variants thereof and polypeptides encoded by the human mahogany sequences.
As used herein, the following terms shall have the abbreviations indicated.
BAC, bacterial artificial chromosomes
bp, base pair(s)
EST, expressed sequence tag
mg, mahogany gene
RFLP, restriction fragment length polymorphism
RT-PCR, reverse transcriptase PCR
SSCP, single-stranded conformational polymorphism
SSLP, simple sequence length polymorphisms
STS, short tag sequence
YAC, yeast artificial chromosome